Synthesis of Isotopically Labeled Flavor Compounds
J. Agric. Food Chem., Vol. 47, No. 7, 1999 2815
Ta ble 1. Ch em ica l a n d Isotop ic P u r ities of th e La beled
Com p ou n d s Syn th esized
(4 N) to dissolve any insoluble residues. The organic phase
was separated from the water phase, and the aqueous solution
a
isotopic purity (%)c
(byproducts in %)c
was then extracted with Et
organic solutions were washed successively with saturated
solutions of NaHCO
(2 × 10 mL) and NaCl (2 × 10 mL) and
then dried over anhydrous Na SO . After removal of the
solvent by evaporation and distillation under vacuum (64-66
C, 10 mbar, B u¨ chi GKR-50, Flawil, Switzerland), 6.84 g (47.5
mmol, 83% yield) of a colorless oil of d -VIII with a purity of
2
O (3 × 50 mL). The combined
labeled
compd
chemical purity in %
(byproducts in %)
b
3
2
d -I
94
6, hexanoic acid)
99
1, Z-isomer)
94
93 ([ H2]-I)
2
4
2
2
(
(5, I; 1, [ H]-I; 1, [ H3]-I)
2
d -II
97 ([ H2]-II)
°
2
(
(3, [ H]-II)
2
d -III
d -IV
99 ([ H2]-III)
9
7% (GC) was obtained: GC RI(DB-5) ) 1170, RI(DB-1701)
2
2
(
3, E,Z- isomer; 3, others) (0.6, [ H]-III, 0.4, [ H3]-III)
2
) 1283, RI(FFAP) ) 1718, RI(DB-Wax) ) 1719; MS-EI 144
(2, M ), 126 (4), 99 (3), 98 (9), 97 (27), 96 (10), 95 (3), 85 (4),
95
96 ([ H2]-IV)
+
2
(
3, E,Z- isomer; 2, others) (4, [ H]-IV)
8
4 (26), 83 (25), 82 (20), 81 (7), 72 (7), 71 (16), 70 (37), 69 (32),
a
Estimation based on GC-FID analysis, see Experimental
Procedures. Presence of trace of solvents (determined by NMR).
Estimation based on GC/MS-PCI analysis, see Experimental
68 (18), 67 (7), 60 (8), 59 (100), 58 (23), 57 (29), 56 (46), 55
b
(
38), 45 (17), 44 (10), 43 (55), 42 (32), 41 (33), 40 (9), 39 (11);
c
+
+
MS-CI (NH
(
3
) 162 (8, [M + NH
4
2
] ), 126 (3, [M - H O] ), 144
Procedures.
+
+
100, M or [M + NH
4
- H
2
O] ).
2
[
2,3- H
2
]-(E)-2-Nonenal (d -II). This could be obtained by
Isotopic purity was calculated from GC/MS data (Rohwed-
oxidation of d -VIII (6.5 g, 45.1 mmol) with PCC using the same
procedure as described for the oxidation of d -VI. Distillation
of the oily residue through a short Vigreux column under
vacuum (45-46 °C, 2 mbar) resulted in 2.72 g (19.2 mmol, 43%
yield) of a colorless oil of d -II. The oil smelled intensely fatty.
Alternatively, d -II was prepared with better yield from d -VIII
der, 1985; Rakoff and Rohwedder, 1992). Clusters of ions
+
+
representing the species from [M + 3] to [M - 2] of both the
deuterated and nonlabeled reference compounds were mea-
sured in the PCI mode on the SSQ 7000 using selected ion
monitoring and isobutane as reactant gas. The nondeuterated
substances were analyzed for isotope correction of the labeled
compounds.
2
by stirring d -VIII (200 mg, 1.4 mmol) with MnO (2.0 g, 23
mmol) in hexane (10 mL) overnight at room temperature. The
suspension was filtered through Celite. The solvent was
evaporated, yielding 150 mg of d -II (1.1 mmol, 76% yield): GC
2
2
Syn th esis of [5,6- H
2 2
]Hexa n a l (d -I). [5,6- H ]Hexan-1-ol
(
d -VI). 5-Hexen-1-ol (V, 8.0 g, 80 mmol) was placed into a
reaction flask containing 250 mL of benzene. Wilkinson’s
catalyst was added at 15 wt % (1.2 g) of the substrate to be
deuterated. The flask was filled (3×) with deuterium gas by
RI(DB-5) ) 1158, RI(DB-1701) ) 1277, RI(FFAP) ) 1529, RI-
+
(DB-Wax) ) 1536; MS-CI (NH
3
) 160 (100, [M + NH
4
] ), 142
+
+
(2, M or [M + NH
4
- H
2
O] ). Chemical and isotopic purities
alternating evacuation of the system and flushing with 2
H
.
are shown in Table 1.
Syn th esis of [3,4- H
[3,4- H
2
2
Pressure was maintained at slightly above atmospheric pres-
sure. The process of deuteration was periodically monitored
by withdrawing a small sample of the solution with a syringe
and analyzing by GC. When deuteration was complete, the
2
]-(E,E)-2,4-Non a d ien a l (d -III) a n d
]-(E,E)-2,4-Decadien al (d-IV). (Z)-1-Methoxy-1-buten-
2
2
3-yne (XI). An aqueous NaOH solution (150 g, 40%) was added
dropwise to an ethanolic solution (80 mL) of IX (30 g, 0.24
mol) and pyridine (1.5 g) to form X. The gas was then purged
from the reaction mixture into a container cooled with dry ice
while passing through two traps, the first trap containing an
solvent was evaporated. Et
deuterated compound and to precipitate the catalyst. After
removal of Et O, 7.0 g (67.3 mmol, 84% yield) of d -VI was
2
O was then added to dissolve the
2
obtained by distillation under vacuum (37-38 °C, 4 mbar) with
2
aqueous NaOH solution (1 M) and the second trap solid CaCl .
a purity of 97% (GC): GC RI(DB-5) ) 875, RI(DB-1701) ) 966,
This step was almost quantitative. A mixture composed of X
(∼11 g, 0.22 mol) and MeONa (2.7 g) in methanol (100 mL)
was heated in an autoclave at 75 °C for 5 h. After cooling,
water (200 mL) was added and the organic phase was
separated from the aqueous layer. The water phase was
RI(FFAP) ) 1350, RI(DB-Wax) ) 1358; MS-EI 86 (25, [M -
+
H
2
O] ), 85 (4), 71 (28), 70 (24), 59 (6), 58 (100), 57 (42), 56
(
25), 55 (25), 45 (25), 44 (30), 43 (28), 42 (27), 41 (10); MS-CI
+
+
(
NH
5,6- H
with a thermometer and a reflux condenser were suspended
4.5 g (67.5 mmol) of PCC and 1.1 g (13.5 mmol) of anhydrous
NaOAc in 130 mL of anhydrous CH Cl . The system was kept
under nitrogen to avoid moisture. d -VI (6.0 g, 57.7 mmol) in
mL of anhydrous CH Cl was added in one portion to the
3
) 139 (15, [M + NH
4
+ NH
3
] ), 122 (100, [M + NH ] ).
4
2
[
2
]Hexanal (d -I). In a 250 mL three-neck flask fitted
treated with an aqueous H
extracted with Et
O (3 × 100 mL). The combined organic layers
were washed with NaHCO
(1 M, 2 × 10 mL) and dried over
Na SO . After removal of the solvent, the residue was distilled
2 4
SO solution (25 mL, 20%) and then
2
1
3
2
2
2
4
under vacuum, yielding 8.9 g (0.11 mol, 50% yield) of a yellow
oil of XI, which polymerized at room temperature: [MS-EI was
identical with that found in the MS library for (Z)-1-methoxy-
1-buten-3-yne] MS-EI 82 (100), 81 (18), 39 (75), 38 (21), 37
5
2
2
magnetically stirred solution cooled at ∼0 °C. The temperature
was then allowed to increase to 15 °C. The reaction was
monitored by GC. When it was finished, 100 mL of anhydrous
1
(12), 50 (30), 53 (28), 51 (20),49 (10), 52 (9); H NMR δ 6.354
Et
black gum. The insoluble residue was washed with anhydrous
Et
2
O was added and the supernatant was separated from the
(dd, J ) 6.5, 0.85 Hz, 1H, 1-CH), 4.519 (dd, J ) 6.5, 2.3 Hz,
1H, 2-CH), 3.800 (s, 3H, OCH ), 3.086 (dd, J ) 2.4, 0.9 Hz,
3
2
O (3 × 20 mL). The combined organic solutions were passed
1H, 4-CH) [The proton NMR data are in good agreement with
those reported by Corey and Albright (1983). The extremely
long spin-lattice relaxation time of the acetylenic proton
demanded a very long relaxation delay (15 min) between the
pulses to permit quantitative integration. The 6.5 Hz coupling
constant between 1-CH and 2-CH indicates Z-configuration
through a short pad of Florisil, and the solvent was removed
by evaporation. Distillation of the oily residue through a short
Vigreux column (30 × 1 cm) under vacuum (21-22 °C, 10
mbar) resulted in ∼2.2 g of d -I (21.6 mmol, 37% yield): GC
RI(DB-5) ) 801, RI(DB-1701) ) 877, RI(FFAP) ) 1063, RI-
+
13
(
(
DB-Wax) ) 1080; MS-CI 137 (25, [M + NH
4
+ NH
3
] ), 120
for the double bond]; C NMR δ 158.08 (d, 1-CH), 84.31 (d,
+
+
+
100, [M + NH
4
] ), 102 (5, M or [M + NH
4
- H
2
O] ). Chemical
3
2-CH), 80.68 (d, 4-CH), 78.25 (s, 3-C), 60.84 (q, OCH ) [These
and isotopic purities are shown in Table 1.
assignments were corroborated by a 13C spectrum without
2
2
2
Syn th esis of [2,3- H
2
]-(E)-2-Non en a l (d -II). [2,3- H
2
]-(E)-
proton decoupling (characteristic acetylenic J CH of ∼50 Hz)
2
-Nonen-1-ol (d -VIII). In a 200 mL three-neck reactor fitted
and for the protonated carbons by a HETCOR experiment].
2
with a reflux condenser and a thermometer was suspended
[3,4- H
2
]-(E,E)-2,4-Nonadienal (d -III). XI (0.75 g, 9.2 mmol),
2
3
.0 g (80 mmol) of LiAl H
4
in anhydrous THF. 2-Nonyn-1-ol
dissolved in anhydrous THF (12 mL), was added dropwise to
a solution of BrMgC (8 mL, 1.0 M in THF) while the
(VII, 8.0 g, 57 mmol) in anhydrous THF (10 mL) was slowly
2 5
H
added to the magnetically stirred solution. The mixture was
refluxed for 1 h under nitrogen and then stored overnight at
mixture was maintained at ∼40 °C. After an additional hour
of stirring at room temperature, the reaction flask was cooled
in an ice-water bath, and a solution of pentanal (0.6 g, 7
mmol) dissolved in anhydrous THF (5 mL) was added drop-
wise. The mixture was stirred at room temperature until the
room temperature. GC analysis indicated complete reduction
2
of VII to d -VIII. After cooling with an ice bath, 20 mL of H
2
O
2 4
was added drop by drop, followed by 80 mL of aqueous H SO